Functional Expression of the Genomic DNA Sequences Encoding Mouse Na,K-ATPase α1 Gene by Cotransfection of Overlapping Genomic DNA Segments

Abstract

The entire 33-kb coding region of the mouse Na,K-ATPase α1 subunit gene was cloned in two overlapping cosmids which contain inserts of 40 kb. To assess the functional expression of the mouse al gene, the two cosmids were cotransfected into ouabain-sensitive CV-1 monkey cells yielding an average of 64 resistant colonies per 10⁶ cells per μg of DNA. Analysis of the DNA transferred to the ouabain-resistant transformants by the two cosmids suggests that the generation of a functional gene can occur by homologous recombination between the two introduced segments, as demonstrated by generation of a novel diagnostic restriction fragment. The ability to reconstruct the intact mouse al gene in a heterologous host cell and to monitor its functional expression with a selection protocol permits direct identification and isolation of regulatory sequences for the gene.